Lipoteichoic Acid Isolated from Weissella Cibaria Increases

J. Microbiol. Biotechnol. (2016), 26(7), 1198–1205 http://dx.doi.org/10.4014/jmb.1601.01047 Research Article Review jmb

Lipoteichoic Acid Isolated from Weissella cibaria Increases Cytokine Production in Human Monocyte-Like THP-1 Cells and Mouse Splenocytes Yi-Fan Hong1†, Yoon-Doo Lee1†, Jae-Yeon Park1, Seongjae Kim1, Youn-Woo Lee1, Boram Jeon1, Deepa Jagdish1, Hangeun Kim1,2, and Dae Kyun Chung1,2,3*

1Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Yongin 17104, Republic of Korea 2Skin Biotechnology Center, Kyung Hee University, Yongin 17104, Republic of Korea 3RNA Inc., #308 College of Life Science, Kyung Hee University, Yongin 17104, Republic of Korea

Received: January 21, 2016 Revised: March 21, 2016 Lactic acid bacteria (LAB) have beneficial effects on intestinal health and skin diseases. Accepted: March 23, 2016 Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is known to induce the production of several cytokines such as TNF-α, IL-1β, and IL-8 and affect the intestinal

First published online microflora, anti-aging, sepsis, and cholesterol level. In this study, Weissella cibaria was isolated March 24, 2016 from Indian dairy products, and we examined its immune-enhancing effects. Live and heat- killed W. cibaria did not induce the secretion of immune-related cytokines, whereas LTA *Corresponding author Phone: +82-31-201-2465; isolated from W. cibaria (cLTA) significantly increased the secretion of TNF-α, IL-1β, and IL-6 Fax: +82-31-202-8333; in a dose-dependent manner. cLTA increased the phosphorylation of nuclear factor kappa- E-mail: [email protected] light-chain-enhancer of activated B cells, p38 mitogen-activated protein kinases, and c-Jun N- †These authors contributed terminal kinases in THP-1 cells. The secretion of TNF-α and IL-6 was also increased in the equally to this work. cLTA-treated mouse splenocytes. These results suggest that cLTA, but not W. cibaria whole pISSN 1017-7825, eISSN 1738-8872 cells, has immune-boosting potential and can be used to treat immunosuppression diseases.

Introduction (pLTA) does not induce TNF-α production. The outstanding function of LTA isolated from L. plantarum is anti- Lipoteichoic acid (LTA) is a cell wall component of gram- inflammatory effects. It has been reported that LTA from positive bacteria. The structure of LTA is different in every L. plantarum inhibits the inflammatory cytokine production species [29]. However, most LTA is composed of long induced by LPS and LTA from S. aureus (aLTA) [12, 13]. phosphate chains and glycolipids. The function of LTA is LTA is recognized by Toll-like receptor 2 (TLR2) [19, 30] similar to that of lipopolysaccharide (LPS), which is a cell and activates nuclear factor kappa-light-chain-enhancer of wall component of gram-negative bacteria. LTA and LPS activated B cells (NF-κB) and mitogen-activated protein increase secretion of pro-inflammatory cytokines such as kinases (MAPK), including p38 mitogen-activated protein TNF, IL-1, and IL-6 [19, 25, 26], which cause inflammatory kinases (p38), c-Jun N-terminal kinases (JNK), and diseases such as arthritis, meningeal inflammation, and extracellular signal-regulated kinases (ERK), which lead to septic shock [6]. However, LTAs from different bacterial the activation of transcription factors that are required for species and strains have different immune regulatory effects. the secretion of inflammatory cytokines [4, 16, 32]. For example, LTA from Bacillus subtilis, Lactobacillus casei, Most lactic acid bacteria (LAB) are well-known probiotics. Lactobacillus fermentem, and Staphylococcus aureus increase LAB prevent the adherence and replication of pathogens the production of TNF-α in RAW 264.7 cells or splenocytes through an antimicrobial system and also effect the [19, 24]. On the other hand, LTA from Lactobacillus plantarum suppression of tumor growth [17, 21]. LAB change the gut

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microflora and thereby affect intestinal health. LAB also with the LAL endotoxin kit (GenScript, NJ, USA). have beneficial effects for reducing alcohol-induced hepatic inflammation and other anti-inflammatory activity [17, 18, Cell Culture 28]. In the food industry, LAB are used to produce fermented THP-1 cells were grown in RPMI 1640 supplemented with 10% foods like kimchi and yogurt. LAB in foods produce some heat-inactivated fetal bovine serum (FBS), and 100 U/ml of penicillin and 100 µg/ml of streptomycin (P/S). They were cultured at 37°C substances that create different tastes or smells and in 5% CO . Male 5-week-old BALB/c mice were obtained from antimicrobial substances like bacteriocins that inhibit the 2 NaraBio (Korea). Mice were housed at 23°C in a 12 h light/dark growth of harmful bacteria [23]. LAB such as L. plantarum, cycle for 7 days. Mouse spleens were isolated and washed with Streptococcus thermophiles, and Bifidobacterium breve are used DPBS. Single-cell suspensions were prepared by grinding the in cosmetic ingredients because they affect skin hydration, spleens into small pieces with a strainer and centrifuging at 800 ×g are antioxidative, and can enhance and produce hyaluronic for 5 min. The cell pellet was resuspended in RBC lysis buffer for acid that affects skin moisturization [9]. Weissella cibaria is a 3 min on ice and centrifuged. After washing in RPMI 1640 gram-positive bacterium the belongs to the Leuconostocaceae medium, the cells were adjusted to a final concentration of 5 × 106 family. Weissella species are one of the most common LAB cells/ml in RPMI 1640 supplemented with 10% FBS and P/S and found in fermented foods, including kimchi [15]. Since cultured at 37°C in 5% CO2. W. cibaria has an inhibitory effect against volatile sulfur compounds and cancer preventive potential, it is considered Enzyme-Linked Immunosorbent (ELISA) Assay THP-1 and splenocytes were seeded at 1 × 106 cells in a 96-well as a novel probiotic [10, 14]. It is also known that W. cibaria round plate. To examine the induction of cytokines by live or inhibits the adherence of Fusobacterim nucleatum to the heat-killed bacteria and LTA, cells were treated for time- and epithelium of the oral cavity by coaggregation with them, dose-dependent studies. To examine the inhibitory effect of LTA, which results in the decrease of bad breath [11]. Previous cells were pretreated with 100 µg/ml LTA for 18 h and then with studies have demonstrated that pLTA has anti-inflammatory 500 ng/ml LPS for 6 h. The secretion of cytokines in the effects [12, 13], and aLTA causes severe inflammation and supernatants was analyzed by sandwich ELISA. hTNF-α, hIL-1β, septic shock [3, 27]. However, the immune-regulating effect hIL-8, mTNF-α, mIL-1β, mIL-6, and mIL-12 were measured using of W. cibaria has not been demonstrated. In this study, we specific antibodies, purchased from R&D Systems (USA). examined the effects of live or heat-killed W. cibaria and LTA isolated from W. cibaria (cLTA) on inflammatory Western Blot Analysis 6 cytokine production, using the human monocyte-like cell THP-1 cells at 1 × 10 cells/ml were seeded in 6-well plates. line THP-1 and mouse splenocytes. After 24 h, cells were treated with cLTA, pLTA, or aLTA for 1 h and then washed twice with DPBS. Cells were lysed with Laemmli loading buffer and boiled at 100°C for 5 min. Proteins Materials and Methods were separated by 12% SDS-PAGE and transferred onto PVDF membranes at 100 V for 1 h. The membranes were blocked with Bacterial Strains 5% skim milk and washed three times with TBST before being W. cibaria was isolated from Indian fermented food. Briefly, incubated with primary antibody overnight. After washing, LAB have been isolated from Dosa, which is the most popular secondary HRP-conjugated antibody was applied for 2 h. Protein fermented product of South India. Dosa was spread on MRS agar bands were detected with ECL, and β-actin was used as a loading plates after serial dilution, and 100 colonies were selected and control. cultured in MRS broth. The cultured LAB samples were numbered CDK 1 to CDK 100. The strain was identified using 16S rRNA Statistical Analysis sequencing, and the phylogenetic tree was drawn. L. plantarum All the experiments were performed at least three times. The was also cultured in MRS broth. S. aureus was cultured in BHI data shown are representative results of the mean ± SD of broth. Cells were harvested by centrifugation at 4,000 ×g for triplicated experiments. Differences were considered statistically 10 min and washed three times with distilled water. significant when the p value was <0.05.

Purification of LTA LTAs from W. cibaria, L. plantarum, and S. aureus were purified Results using the methods of Morath et al. [20]. Briefly, harvested cells W. cibaria Was Isolated from Dosa, a Traditional Indian were sonicated and then extracted with butanol. Octyl-Sepharose Fermented Food and DEAE Sepharose chromatography were used to purify LTAs. The purity of the LTAs was determined by measuring the protein CDK18 was isolated from an Indian fermented food as content with silver staining, and endotoxin contents were identified described in Materials and Methods. A single colony of

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bacteria was sequenced for phylogenetic analysis. The 16S S. aureus, which significantly increased TNF-α production rRNA gene was sequenced, and sequence alignment was after 24 h stimulation (Fig. 2A). TNF-α secretion was not performed using NCBI BLAST (http://blast.ncbi.nlm.nih.gov). altered in heat-killed W. cibaria-treated cells, whereas it was A neighbor-joining tree based on 16S rRNA gene sequences significantly increased by heat-killed S. aureus (Fig. 2B). showed the phylogenetic relationships between Weissella Live and heat-killed S. aureus-treated cells significantly and CDK18, with a Bar 0.02 substitutions per nucleotide increased TNF-α after 3 h stimulation. Similarly, time- position (Fig. 1). Bootstrap analysis with 1,000 replicates dependent induction of TNF-α was shown in live and heat- was also conducted in order to obtain confidence levels for killed W. cibaria-treated cells, but it was not significant the branches. Most of the species in Weissella were included (Figs. 2C and 2D). These results indicate that live and heat- in the phylogenetic tree. CDK18 was identified as W. cibaria killed W. cibaria do not affect immune responses. and renamed W. cibaria K401 for further studies. Next, the biological function of cLTA was examined. To examine the cytotoxicity of cLTA, the viability of cLTA- cLTA Isolated from W. cibaria Induced Cytokine Production treated THP-1 cells was identified using the WST-1 assay. in THP-1 Cells cLTA, pLTA, and aLTA did not induce cell death, which To examine the effects of W. cibaria on cytokine production, indicates that cLTA does not affect the viability of THP-1 live and heat-killed W. cibaria were applied dose- and time- cells (Fig. 3A). To investigate the effects of cLTA on cytokine dependently. TNF-α secretion was increased to 1 × 108 production, culture supernatants from cLTA-treated THP-1 CFU/ml, but the production level was less than that of cells were collected and subjected to ELISA. As shown in

Fig. 1. Phylogenetic tree of CDK18. A single colony of CDK18, isolated from an Indian fermented food, was sequenced for its 16S rRNA. The data show that CDK18 is closely related to Weissella cibaria. Thus, we renamed CDK18 as W. cibaria K401.

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Fig. 2. Dose-dependent (A, B) and time-dependent (C, D) effects of live and heat-killed W. cibaria, L. plantarum, and S. aureus on TNF-α secretion. THP-1 cells were seeded at 1 × 106 cells in a 96-well round plate with antibiotic-free medium. Live (A) and heat-killed (B) bacteria were treated at the indicated doses. After 6 h, culture supernatants were collected and the TNF-α level was measured using ELISA. (C, D) Levels of TNF-α by live and heat-killed W. cibaria, L. plantarum, and S. aureus (1 × 108 CFU/ml) in a time-dependent manner. Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated cells.

Fig. 3, cLTA significantly induced TNF-α, IL-1β, and IL-8 shown in Fig. 4, the level of p-ERK was not changed by production compared with untreated cells (Figs. 3B, 3C, LTA. However, the levels of p-JNK, p-p38, and NF-κB were and 3D). Unlike whole-cell bacteria, cLTA seems to have a significantly increased by cLTA. Similar results were strong immune regulatory effect. Previous studies have shown in aLTA-treated cells, whereas pLTA did not alter shown that pLTA isolated from L. plantarum inhibited signaling variation. These results indicate that cLTA excessive inflammation caused by LPS [12]. To examine the induces the production of inflammatory cytokines by inhibitory effects of cLTA, LPS-mediated TNF-α production activating the JNK, p38, and NF-κB signaling pathways. was examined after pretreatment with cLTA. Unexpectedly, cLTA pretreatment did not inhibit LPS-mediated TNF-α cLTA Induces Inflammatory Cytokines in Mouse Splenocytes production (Fig. 3E). LPS-mediated IL-1β and IL-8 production When mouse splenocytes were treated with cLTA, unlike was also not changed by cLTA pretreatment (data not shown). with THP-1 cells, the secretion of TNF-α from splenocytes was increased by all LTAs used in this study (Fig. 5A), cLTA Increased the Phosphorylation of NF-κB and MAPK including cLTA. Interestingly, the results of production of in THP-1 Cells TNF-α by aLTA at 100 µg/ml and 10 µg/ml were similar. Previous studies have shown that activation of MAPK We did not examine the toxicity of LTA on splenocytes, but and NF-κB signals is important to produce inflammatory it seems that splenocytes were killed by aLTA at 100 µg/ cytokines [4, 16, 32]. Thus, to identify the effect of cLTA on ml, because aLTA is toxic to cells and causes shock and signaling activation, western blot assay was performed to organ failure [3]. The secretion of IL-6, like that of TNF-α, measure the activation of ERK, JNK, p38, and NF-κB. As was also increased by LTAs (Fig. 5B). The cLTA-mediated

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Fig. 3. Effects of cLTA, pLTA, and aLTA on cytokine production in THP-1 cells. THP-1 cells were seeded at 1 × 104 cells in a 96-well round plate. Then, the cells were cultured with LTAs at 1-100 µg/ml for 24 h. Cell viability was measured by WST-1 assay. The results were normalized with None (A). THP-1 cells were cultured with the indicated concentrations of LTAs for 6 h. Culture supernatants were collected, and TNF-α (B), IL-1β (C), and IL-8 (D) levels were determined using ELISA. THP-1 cells were pretreated dose-dependently with LTAs for 18 h, and LPS was applied at 500 ng/ml for 6 h. Then, the supernatants were collected and TNF-α levels were measured using ELISA (E). Data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated or LPS- treated only cells. production of IL-6 was higher than that of pLTA, but this treated splenocytes. Pretreatment with cLTA did not was not significant compared with aLTA. IL-1β and IL-12 inhibit the production of TNF-α induced by LPS (Fig. 5C). were not increased by LTA (data not shown). Like THP-1 cells, it seems that cLTA is involved in the Next, we examined the inhibitory effects of cLTA on LPS- TNF-α pathway, but not in the inhibition pathway. IL-6

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Fig. 4. Activation of MAPK and NF-κB signaling by cLTA, pLTA, and aLTA in THP-1 cells. THP-1 cells were cultured with 100 µg/ml LTAs for 1 h. The levels of p-JNK, p-ERK, p-p38, and NF-κBp65 (Cell Signaling Technology, MA, USA) were measured using a western blot assay and normalized to β- actin. and IL-1β were increased by LPS, but they were not inhibited by any LTA (data not shown).

Discussion

Most LAB are probiotics that have many beneficial effects on immune and intestinal regulation. LTA has effects on aging, skin whitening, and modulating inflammatory cytokines [12, 13]. Our study focused on the immune- regulating effect of W. cibaria. The effects of live and heat- killed W. cibaria on the production of inflammatory cytokines were very weak. However, cLTA showed immune- stimulating effects. Inflammatory cytokines, such as TNF- α, IL-1β, and IL-8, were increased by cLTA, which also activated the phosphorylation of NF-κB and MAPK. These inducing effects by cLTA were shown on THP-1 cells, so we performed these experiments with mouse splenocytes. The inducing effects of cLTA were also shown in mouse Fig. 5. Effects of cLTA, pLTA, and aLTA on the production of splenocytes, and cLTA increased TNF-α and IL-6 in a dose- cytokines in splenocytes. dependent manner. However, the phosphorylation of ERK Pure splenocytes were seeded at 1 × 106 cells in a 96-well round plate. was decreased in cLTA-treated THP-1 cells. The activation After 1 day, cLTA, pLTA, and aLTA were treated at the indicated of MAPK induces different cell signaling. For example, concentrations for 6 h. Culture supernatants were collected, and the JNK plays a role in T cell differentiation and the cellular levels of TNF-α (A) and IL-6 (B) were determined using ELISA. (C) apoptosis pathway. The activation of this signaling pathway Splenocytes were cultured with the indicated concentrations of LTAs for 18 h and stimulated with 500 ng/ml LPS for 6 h. The supernatants contributes to inflammatory responses in mammals [8]. P38 were collected, and TNF-α levels were measured using ELISA. Data MAPK, which is activated by inflammatory cytokines, LPS, are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 ultraviolet light (UV), and growth factors, is involved in compared with untreated or LPS-treated only cells.

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cell differentiation, apoptosis, and autophagy [7]. ERK is differences of LTA. For example, Streptococcus pneumoniae activated by many stimuli, including cytokines, viruses, R6 and S. pneumoniae Fp23 are different strains of S. pneumoniae carcinogens, and growth factors, and is involved in the that have different structures of LTA and immunostimulatory regulation of meiosis, mitosis, and post-mitotic functions in potencies [5]. aLTA also stimulates an immune response, differentiated cells [2]. Interestingly, aLTA and pLTA did but has other side effects such as inducing sepsis and other not alter the ERK phosphorylation, whereas cLTA slightly diseases. However, W. cibaria is one LAB that is harmless decreased it, indicating that the ERK signaling pathway and well known for its probiotic effects. Thus, it can be may not be altered by LTA-TLR2 stimulation. Although the used in the treatment of immune-compromised patients. phosphorylation of ERK by cLTA decreased in THP-1 cells, Inflammation is a double-edged sword. Cytokine induction we do not expect that decreased ERK phosphorylation is required for the activation of the immune system, but affects cell states, including cytokine production, since over-activation leads to the inflammatory diseases. Thus, cells were activated by other signaling pathways. After the fine regulation of inflammation is important to maintain stimulation by LTA, the activation of signaling molecules, homeostasis. Any components from bacteria can be used for including NF-κB and MAPKs, begins within 15 min and therapies if they are highly purified and do not induce any returns to the steady state in 2 h. The activated signaling side effects. Commercially available monophosphoryl lipid A molecules induce the transcription and translation of certain (MPLA), for example, is extracted from lipopolysaccharide genes. The mRNA level of pro-inflammatory cytokines, for (LPS or endotoxin) produced by Escherichia coli or Salmonella example, reaches a peak at 2 to 3 h and the protein level Minnesota [1]. MPLA is a TLR4 agonist that is currently reaches a peak at 3 to 4 h. After reaching a peak, cytokine used as a vaccine adjuvant in humans, although it is production is returned to the steady-state level. However, developed from pathogens. Cytokine-inducible LTAs, TNF-α production in the bacteria-treated THP-1 cells was including cLTA and aLTA may be applied to induce maintained up to 24 h. It might be because whole-cell inflammation in immunosuppressed patients, but they bacteria have plenty of ligands to stimulate immune cells should not be applied to patients who have systemic on their surface. In this experiment, induction was not inflammatory response syndrome including sepsis. shown in live or heat-killed W. cibaria. Many studies have In conclusion, this study demonstrated the immune- shown different results between live and heat-killed bacteria boosting effects of cLTA. cLTA induces the production of on stimulation of the immune response. For example, live inflammatory cytokines such as TNF-α by activating the L. reuteri has anti-inflammatory effects, but heat-killed and phosphorylation of NF-κB and MAPK. These results gamma-irradiated bacteria do not have these effects [18]. suggest that cLTA has treatment potential in immune On the other hand, both live and heat-killed L. paracasei suppression. However, further studies are needed to verify have effects on perennial allergic rhinitis [22]. It seems that the effect of cLTA on treatment of immunosuppressed the similar effects of live and heat-killed bacteria are due to patients. the cell wall component. In this study, live and heat-killed W. cibaria showed Acknowledgments similar moderate cytokine production, indicating that cell wall components are involved in the immune regulation. In This work was supported by a grant from Kyung Hee the study using LTA, one of the gram-positive bacteria cell University in 2011 (KHU-20110263). wall components, cLTA, showed cytokine-inducing effects, unlike live and heat-killed W. cibaria. LTA of various bacteria References have different immunostimulatory effects [24]. Previous studies have shown that different LTAs have different 1. Aybay C, Imir T. 1998. Comparison of the effects of Salmonella minnesota Re595 lipopolysaccharide, lipid A and effects. For example, aLTA increases the production of monophosphoryl lipid A on nitric oxide, TNF-alpha, and IL- TNF-α and induces organ failure [3]. LTA isolated from 6 induction from RAW 264.7 macrophages. FEMS Immunol. L. sakei (sLTA) induces TNF-α production, but sLTA can Med. Microbiol. 22: 263-273. inhibit the production of TNF-α induced by LPS [31]. pLTA 2. Boulton TG, Cobb MH. 1991. Identification of multiple does not increase TNF-α production and inhibits TNF-α extracellular signal-regulated kinases (ERKs) with antipeptide induced by aLTA and LPS [12, 13]. cLTA induced the antibodies. Cell Regul. 2: 357-371. inflammatory cytokine but did not inhibit the cytokines 3. De Kimpe SJ, Kengatharan M, Thiemermann C, Vane JR. induced by LPS. This effect seems to be due to structural 1995. The cell wall components peptidoglycan and lipoteichoic

J. Microbiol. Biotechnol. W. cibaria LTA Enhances Immune Responses 1205

acid from Staphylococcus aureus act in synergy to cause Ishikawa K, Yokokura T, Yoshikai Y. 2003. Lipoteichoic acid shock and multiple organ failure. Proc. Natl. Acad. Sci. USA from Lactobacillus strains elicit strong tumor necrosis factor 92: 10359-10363. alpha-inducing activities in macrophages through Toll-like 4. Dong C, Davis RJ, Flavell RA. 2002. MAP kinases in the receptor 2. Clin. Diagn. Lab. Immunol. 10: 259-266. immune response. Annu. Rev. Immunol. 20: 55-72. 20. Morath S, Geyer A, Hartung T. 2001. Structure-function 5. Draing C, Pfitzenmaier M, Zummo S, Mancuso G, Geyer A, relationship of cytokine induction by lipoteichoic acid from Hartung T, von Aulock S. 2006. Comparison of lipoteichoic Staphylococcus aureus. J. Exp. Med. 193: 393-397. acid from different serotypes of Streptococcus pneumoniae. J. 21. Naidu AS, Bidlack WR, Clemens RA. 1999. Probiotic spectra of Biol. Chem. 281: 33849-33859. lactic acid bacteria (LAB). Crit. Rev. Food Sci. Nutr. 39: 13-128. 6. Ginsburg I. 2002. Role of lipoteichoic acid in infection and 22. Peng GC, Hsu CH. 2005. The efficacy and safety of heat- inflammation. Lancet Infect. Dis. 2: 171-179. killed Lactobacillus paracasei for treatment of perennial allergic 7. Han J, Lee JD, Bibbs L, Ulevitch RJ. 1994. A MAP kinase rhinitis induced by house-dust mite. Pediatr. Allergy Immunol. targeted by endotoxin and hyperosmolarity in mammalian 16: 433-438. cells. Science 265: 808-811. 23. Rhee SJ, Lee JE, Lee CH. 2011. Importance of lactic acid 8. Ip YT, Davis RJ. 1998. Signal transduction by the c-Jun N- bacteria in Asian fermented foods. Microb. Cell Fact. 10: S5. terminal kinase (JNK) - from inflammation to development. 24. Ryu YH, Baik JE, Yang JS, Kang SS, Im J, Yun CH, et al. Curr. Opin. Cell Biol. 10: 205-219. 2009. Differential immunostimulatory effects of gram-positive 9. Izawa N, Sone T. 2014. Cosmetic ingredients fermented by bacteria due to their lipoteichoic acids. Int. Immunopharmacol. lactic acid bacteria. Microb. Prod. 233-242. 9: 127-133. 10. Kang MS, Kim BG, Chung J, Lee HC, Oh JS. 2006. Inhibitory 25. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning effect of Weissella cibaria isolates on the production of CJ. 1999. Peptidoglycan- and lipoteichoic acid-induced cell volatile sulphur compounds. J. Clin. Periodontol. 33: 226-232. activation is mediated by Toll-like receptor 2. J. Biol. Chem. 11.Kang MS, Na HS, Oh JS. 2005. Coaggregation ability of 274: 17406-17409. Weissella cibaria isolates with Fusobacterium nucleatum and 26. Su SC, Hua KF, Lee H, Chao LK, Tan SK, Lee H, et al. 2006. their adhesiveness to epithelial cells. FEMS Microbiol. Lett. LTA and LPS mediated activation of protein kinases in the 253: 323-329. regulation of inflammatory cytokines expression in macrophages. 12. Kim HG, Kim NR, Gim MG, Lee JM, Lee SY, Ko MY, et al. Clin. Chim. Acta 374: 106-115. 2008. Lipoteichoic acid isolated from Lactobacillus plantarum 27. Wang JE, Jørgensen PF, Almlöf M, Thiemermann C, Foster inhibits lipopolysaccharide-induced TNF-alpha production SJ, Aasen AO, Solberg R. 2000. Peptidoglycan and lipoteichoic in THP-1 cells and endotoxin shock in mice. J. Immunol. 180: acid from Staphylococcus aureus induce tumor necrosis factor 2553-2561. alpha, interleukin 6 (IL-6), and IL-10 production in both T 13. Kim HG, Lee SY, Kim NR, Ko MY, Lee JM, Yi TH, et al. 2008. cells and monocytes in a human whole blood model. Infect. Inhibitory effects of Lactobacillus plantarum lipoteichoic acid Immun. 68: 3965-3970. (LTA) on Staphylococcus aureus LTA-induced tumor necrosis 28. Wang Y, Liu Y, Kirpich I, Ma Z, Wang C, Zhang M, et al. factor-alpha production. J. Microbiol. Biotechnol. 18: 1191-1196. 2013. Lactobacillus rhamnosus GG reduces hepatic TNFα 14. Kwak SH, Cho YM, Noh GM, Om AS. 2014. Cancer production and inflammation in chronic alcohol-induced preventive potential of kimchi lactic acid bacteria (Weissella liver injury. J. Nutr. Biochem. 24: 1609-1615. cibaria, Lactobacillus plantarum). J. Cancer Prev. 19: 253-258. 29. Wicken AJ, Knox KW. 1975. Lipoteichoic acid: a new class 15.Lee KW, Park JY, Chun J, Han NS, Kim JH. 2010. of bacterial antigen. Science 187: 1161-1167. Importance of Weissella species during kimchi fermentation 30. Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, and future works. Kor. J. Microbiol. Biotechnol. 38: 341-348. Golenbock D. 1999. Cutting edge: recognition of gram- 16. Lehner MD, Morath S, Michelsen KS, Schumann RR, Hartung positive bacterial cell wall components by the innate T. 2001. Induction of cross-tolerance by lipopolysaccharide immune system occurs via Toll-like receptor 2. J. Immunol. and highly purified lipoteichoic acid via different Toll-like 163: 1-5. receptors independent of paracrine mediators. J. Immunol. 31. You GE, Jung BJ, Kim HR, Kim HG, Kim TR, Chung DK. 166: 5161-5167. 2013. Lactobacillus sakei lipoteichoic acid inhibits MMP-1 17. Ljungh A, Wadström T. 2006. Lactic acid bacteria as induced by UVA in normal dermal fibroblasts of human. J. probiotics. Curr. Issues Intest. Microbiol. 7: 73-89. Microbiol. Biotechnol. 23: 1357-1364. 18. Ma D, Forsythe P, Bienenstock J. 2004. Live Lactobacillus 32. Zbinden-Foncea H, Raymackers JM, Deldicque L, Renard P, reuteri is essential for the inhibitory effect on tumor necrosis Francaux M. 2012. TLR2 and TLR4 activate p38 MAPK and factor alpha-induced interleukin-8 expression. Infect. Immun. JNK during endurance exercise in skeletal muscle. Med. Sci. 72: 5308-5314. Sports Exerc. 44: 1463-1472. 19. Matsuguchi T, Takagi A, Matsuzaki T, Nagaoka M,

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